Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/34—Allele or polymorphism specific uses

Definitions

• MYC is a proto-oncogene that is overexpressed in many human cancers. It plays an important role in many biological pathways related to neoplastic cell growth and proliferation. New and effective ways are required to target Myc for cancer therapeutics.
• compositions comprising a synthetic nucleic acid sequence encoding a Plasmacytoma variant translocation 1 217 (PVT 1 217) splice variant micropeptide, comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1.
• the PVT1 217 splice variant micropeptide is at least 12 amino acids in length.
• the PVT 1 217 splice variant micropeptide is at least 13 amino acids in length.
• the PVT 1 217 splice variant micropeptide is at least 14 amino acids in length.
• compositions comprising a synthetic nucleic acid sequence encoding a Plasmacytoma variant translocation 1 217 (PVT1 217) splice variant micropeptide, comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1, wherein the PVT1 217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1 217.
• PVT1 217 Plasmacytoma variant translocation 1 217
• shORF short open reading frame
• the PVT1 217 splice variant micropeptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT 1 217.
• the nucleic acid is DNA.
• the nucleic acid is a messenger RNA.
• the synthetic nucleic acid comprises one or more modified nucleotides.
• the synthetic nucleic acid sequence encoding a PVT1 217 splice variant micropeptide is comprised in a vector.
• vectors comprising: a nucleic acid sequence encoding a PVT1 217 splice variant micropeptide, wherein the PVT1 217 splice variant micropeptide is at least 12 amino acids in length, and comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1 217.
• the nucleic acid sequence encoding a PVT 1 217 splice variant micropeptide has least 80% sequence identity to the sequence set forth in SEQ ID NO: 1.
• the vector comprises self- replicating RNA vector.
• compositions comprising: (i) a synthetic nucleic acid sequence encoding a Plasmacytoma variant translocation 1 217 (PVT1 217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1; and (ii) a pharmaceutically acceptable excipient.
• the PVT1 217 splice variant micropeptide is at least 12 amino acids in length.
• the PVT1 217 splice variant micropeptide is at least 13 amino acids in length.
• the PVT 1 217 splice variant micropeptide is at least 14 amino acids in length.
• the PVT1 217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1 217. In some embodiments, the PVT1 217 splice variant micropeptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1 217.
• the synthetic nucleic acid sequence encoding the PVT1 217 splice variant micropeptide has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 1.
• the synthetic nucleic acid is DNA.
• the synthetic nucleic acid is a messenger RNA.
• the synthetic nucleic acid comprises one or more modified nucleic acids.
• the synthetic nucleic acid sequence encoding a PVT1 217 splice variant micropeptide is comprised in a vector.
• the vector is a mammalian expression vector.
• the vector is a lentiviral expression vector.
• the vector comprises a promoter.
• the promoter is inducible.
• the pharmaceutical compositions disclosed herein further comprise a cancer cell targeting moiety.
• a pharmaceutical composition comprising a nucleic acid sequence encoding a PVT1 217 splice variant micropeptide, comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1.
• the nucleic acid sequence encoding the PVT1 217 splice variant micropeptide comprises a sequence set forth in SEQ ID NO: 1.
• the PVT1 217 splice variant micropeptide comprises 14 amino acids.
• the PVT 1 217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1 217.
• the pharmaceutical composition results in reduction in cancer cell division.
• an isolated peptide comprising an amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80% identity to SEQ ID NO: l.
• the isolated peptide may comprise the amino acid sequence is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.
• the amino acid sequence is at least 12 amino acids in length. In some embodiments, the amino acid sequence is at least 13 amino acids in length. In some embodiments, the amino acid sequence is at least 14 amino acids in length. In some embodiments, the the peptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at a junction of Exon 3 and Exon 4 of human PVT1 217. In some embodiments, the the peptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT 1 217.
• shORF short open reading frame
• the amino acid sequence comprises at least 1 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 2 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 3 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 4 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence comprises at least 5 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the isolated peptide has an amino acid sequence of SEQ ID NO: 1.
• a pharmaceutical composition comprising: a Plasmacytoma variant translocation 1 217 (PVT1 217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1; and, a pharmaceutically acceptable excipient.
• PVT1 217 Plasmacytoma variant translocation 1 217
• splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1
• a pharmaceutically acceptable excipient comprising: a Plasmacytoma variant translocation 1 217 (PVT1 217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1; and, a pharmaceutically acceptable excipient.
• the PVT1 217 splice variant micropeptide is at least 12 amino acids in length. In some embodiments, the PVT1 217 splice variant micropeptide is at least 13 amino acids in length. In some embodiments, the PVT 1 217 splice variant micropeptide is at least 14 amino acids in length. [0011] In some embodiments, the PVT1 217 splice variant micropeptide comprises at least 10 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1 217.
• shORF short open reading frame
• the PVT1 217 splice variant micropeptide comprises a maximum of 14 contiguous amino acids that are identical to a peptide encoded by a short open reading frame (shORF) located at the junction of Exon 3 and Exon 4 of human PVT1 217.
• shORF short open reading frame
• the micropeptide is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.
• the micropeptide has a sequence of SEQ ID NO: 1.
• the micropeptide comprises at least 1 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 2 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 3 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 4 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 5 conservative amino acid substitution within the sequence of SEQ ID NO: 1.
• the micropeptide is further conjugated with one or more biomolecules.
• the biomolecule is a peptide.
• the biomolecule is a lipid.
• the micropeptide is further modified.
• the modification is selected from the group consisting of: myristoylation, palmitoylation, isoprenylation, glypiation, lipolation, acylation, alkylation, amidation, phosphorylation, glycation, biotinylation, pegylation, sumoylation, ubiquitination, neddylation, or pupylation.
• the pharmaceutical composition comprises a recombinant protein comprising the micropeptide described herein, having at least 80% sequence identity to SEQ ID NO: 1.
• the peptide is associated with a carrier molecule.
• the carrier molecule is a lipid.
• the pharmaceutical composition described herein is for use in preparing a medicament for the treatment of a cancer in a subject.
• a method of treating cancer in a subject in need thereof comprising: administering to the subject a pharmaceutical composition comprising a micropeptide having a sequence of SEQ ID NO: 1, or a sequence that is at least 80% identical to SEQ ID NO: 1.
• the cancer is a MYC-driven cancer.
• composition comprising a Plasmacytoma variant translocation 1 217 (PVT1 217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1 in the treatment of a cancer in a subject.
• PVT1 217 Plasmacytoma variant translocation 1 217
• splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1 in the treatment of a cancer in a subject.
• composition comprising a nucleic acid encoding Plasmacytoma variant translocation 1 217 (PVT1 217) splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1 in the treatment of a cancer in a subject.
• PVT1 217 Plasmacytoma variant translocation 1 217
• splice variant micropeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1 in the treatment of a cancer in a subject.
• FIG. l is a data from a western blot depicting the effect of siRNA E9 on c-Myc protein expression level in the MSTO-211H cell line (from left to right: si -Ctrl, si-C2, and si-E9).
• FIG. 2 is a graphical representation of chromosomal maps of PVT 1 region showing exons ofPVTl_217 splice variants.
• FIG. 3 is a graphic representation of the various PVT1 splice variants (Source: Ensembl database). Splice variant PVT1_212 and PVT1_217 are highlighted.
• FIG. 4A is a graphic representation of the four exons of PVTl ts , also indicating that siRNA si_E9 targets the sequence on Exon 4.
• FIG. 4B is representative data from exemplary studies showing proliferation in eight Hi- and Lo-MYC cell lines transfected with si-E9 siRNA or control siRNA.
• FIG. 5A-5D depicts western blot analysis of MYC protein in Hi- and Lo-MYC cell lines transfected with si Ctrl (blue left-hand column in each bar graph) and si_E9 (green right-hand column in each bar graph). An antibody against Actin was used as a control to quantify relative MYC expression in the treated cells.
• FIG. 6A depicts a schematic representation of the short open reading frame (shORF) at the junction of Exon 3-4 of PVTl ts and the 14 amino acids that the shORF encodes. Below the schematic representation is the alignment of the amino acid sequence of micropeptide HNB (lclquery_280280) with other similar peptide sequences, using Clustal Omega. The conservation index is shown below the alignments.
• FIG. 6B depicts phylogenetic analysis of the peptide sequences showing similarity with HNB (lclquery_280280).
• FIG. 7A-7B shows representative photomicrographs depicting levels of cell proliferation of Hi- and Lo-MYC cell lines transfected with GFP, HNB or HNB(ATG>TGA) constructs.
• FIG. 8 shows flow cytometry data for verification of a Doxycyclin inducible lentiviral system containing GFP as a transgene.
• FIG. 9A-B depict representative data indicating the effect of HNB expression on cell proliferation in Doxycyclin-inducible GFP expressing transduced cell lines MSTO-211H-HNB 1 (FIG. 9A) and U20S-HNB 1 (FIG. 9B).
• FIG. 9C depicts western blot analysis of c-MYC expression relative to b-Actin expression as control showing induction of HNB expression results in reduced MYC protein.
• FIG. 10 depicts the predicted structure of the 14 amino acid micro-peptide HNB using Swiss-Pdb viewer, anterior view (left), posterior view (middle) and electron density map (web) view of HNB.
• FIG. 11A-B depicts a predicted molecular interaction between KRas and HNB.
• FIG. 11A (left) depicts the crystal structure of the human KRas dimer (id 4TQA in protein database),
• FIG. 11B (right) depicts the molecular docking of HNB (yellow, in center) between the KRas dimers.
• FIG. 12 depicts the predicted disruption of KRas (homodimer shown in left-hand panel) by the HNB micropeptide (shown in red, right-hand panel).
• FIG. 13 depicts results from analysis of CCLE dataset and indicates that PVT1 is the most frequent participant in gene fusions in the CCLE dataset.
• FIG. 14 depicts PVT1 fusion positive cancer types. Different types of PVT 1 fusion positive cancers represented in the CCLE dataset are shown in the Donut plot with the slice proportional to the numbers for each indicated cancer type at the bottom [0037]
• FIG. 15 depicts diverse partners of PVT1. All the different partners of PVT1 are shown in the Sankey plot, with the thickness of the connecting ribbon representing the number of times each fusion is observed in CCLE.
• FIG. 16 depicts Circos plot showing inter and intrachromosomal partners of PVT E
• FIG. 17 depicts distinct types of translocations in PVT1 locus.
• FIG. 18 depicts western blot of MYC in cell lines with breakpoints in PVT1 locus (Hi- MYC) and cell lines without breakpoints in PVT1.
• FIG. 19 depicts whole genome sequencing for COLO-320DM, SK-PN-DW and D458 showing genomic enrichment of 5’ end of PVT 1 with concurrent depletion of the 3’ end of PVT1.
• FIG. 20A and 20B depicts mapping and validation of the PVT1 junction points in the ecDNA from COLO-320DM and D458.
• Myc avian myelocytomatosis viral oncogene homolog
• Myc is a transcription factor encoded by the oncogene MYC, and is associated with various cancers. It is estimated to contribute to at least 75% of all human cancers, including prostate, breast, colon and cervical cancers, myeloid leukemia, lymphomas, small-cell lung carcinomas, and neuroblastoma, among others. High expression of Myc can drive tumorigenesis in several tissue types. Myc is also associated with treatment resistant and lethal outcomes.
• the present disclosure is related to a novel finding that Myc driven cell proliferative function can be inhibited by the micropeptides disclosed and described herein, and that the micropeptides can be used for therapeutic intervention in Myc- associated cancers.
• the terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker).
• a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker.
• the term “gene,” as used herein, refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory regions such as promoters, operators, terminators and the like, which may be located upstream or downstream of the coding sequence.
• polypeptide encompasses amino acid molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to chemically modified amino acids such as amino acid analogs, naturally occurring non-proteogenic amino acids such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid.
• the at least one modified or unusual amino acid is selected from the group consisting of 2-aminoadipic acid; 3-aminoadipic acid; beta-alanine, beta-amino- propionic acid; 2-aminobutyric acid; 4-aminobutyric acid, piperidinic acid; 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2’-diaminopimelic acid; 2,3-diaminopropionic acid; N- ethylasparagine; hydroxylysine; allo-hydroxyline; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine, sarcosine; N-methylisoleucine; 6-N- methyllysine; N-methylvaline
• polypeptide also encompass amino acid sequence variants of a protein or peptide.
• Amino acid sequence variants of the HNB polypeptides disclosed herein can be substitutional, insertional or deletion variants.
• Deletion variants lack one or more residues of the native protein that are not essential for function or immunogenic activity, as exemplified by variants of integral membrane proteins that lack a transmembrane sequence.
• Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide.
• substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the polypeptide, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
• Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
• sequences that have between about 70% and about 80%, or between about 81% and 90% or between about 91% and about 99% of amino acids that are identical or functionally equivalent to the amino acids of the HNB polypeptides disclosed herein are considered biologically functionally equivalent, provided the biological activity of the HNB polypeptide is maintained, and within the scope of the HNB polypeptides disclosed herein.
• the terms “treat,” “treating,” and “treatment” is meant to include alleviating or abrogating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.
• Desirable effects of treatment can include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state and remission or improved prognosis.
• terapéuticaally effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated.
• therapeutically effective amount also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician.
• MYC-driven cancer refers to a cancer characterized by aberrant (typically augmented expression) expression of the c-MYC gene or the Myc protein.
• an individual receiving therapy comprising the HNB polypeptides disclosed herein may be identified as having a susceptibility to a cancer therapy, including e.g., a MYC- dependent susceptibility to a cancer therapy or a KRas-dependent susceptibility to a cancer therapy.
• a MYC-driven neoplasm having a MYC-dependent susceptibility to a cancer therapy may be more susceptible to the cancer therapy than the corresponding neoplasm that lacks or displays reduced MYC expression (including e.g., where the MYC expression is conditionally controlled).
• a KRas-driven neoplasm having a KRas-dependent susceptibility to a cancer therapy may be more susceptible to the cancer therapy than the corresponding neoplasm that lacks or displays reduced KRas expression (including e.g., where the KRas expression is conditionally controlled).
• “Cancer therapy” as used herein, refers to any cancer therapy including but not limited to e.g., radiation therapy, chemotherapy, immunotherapy, and the like.
• Myc tightly regulates a broad set of genes essential to growth and proliferation.
• MYC is also tightly regulated at transcriptional, translational and post-translational levels.
• Myc levels are controlled by multiple mechanisms, including negative autoregulation, gene expression, mRNA, and protein stability and degradation, which all become deregulated in human cancers.
• the MYC gene is found at locus 8q24.21 in a broader region on chromosome 8, which is frequently amplified in cancers. Its two paralogs, N-Myc and L-Myc, which are encoded by MYCN and MYCL genes, were respectively identified in neuroblastoma and lung cancer as tissue-specific factors.
• Human Myc contains several highly conserved regions that are functionally important and are organized in the same fashion among the three Myc paralogs, including: a largely unstructured N-terminal transactivation domain (TAD) and an intrinsically disordered C-terminal region comprising the basic, helix-loop-helix, leucine zipper (bHLHLZ) dimerization, and DNA-binding domains.
• TAD N-terminal transactivation domain
• bHLHLZ leucine zipper
• Mechanisms that account for Myc deregulation include: amplifications or chromosomal translocations of the MYC locus that provoke its exacerbated expression, MYC mRNA destabilization through both direct and indirect regulatory events, and alteration in Myc protein turnover rate. The latter is due to either alterations in Myc protein stability normally dependent on Myc’s phosphorylation status but caused by mutations in key phosphorylation sites or alterations of expression of proteins that are involved in Myc’s post-translational modifications.
• the human MYC gene is approximately 6 kilobases long. It contains three exons: a large non-coding exon 1, followed by coding exons 2 and 3. There are four distinct promoters, Po, Pi, P 2 , and P3 that drive MYC transcription. There are two major translation start codons (CTG, and ATG), from which two universally expressed Myc proteins arise, and there are two polyadenylation signals and several DNAse 1 -hypersensitive sites. Po transcripts start at multiple initiation sites. Pi and P 2 are the two major classical TATA-containing promoter start sites located at the 5' end of exon 1, with greater than three-quarters of MYC transcripts originating from the P 2 promoter. The MYC promoter region is regulated by a large number of signaling pathways, transcription factors, cis-regulatory elements, chromatin remodeling, and by its auto suppression.
• Plasmacytoma variant translocation 1 is a Tong non-coding RNA’ transcribed from adjacent to the oncogene c-MYC, and has been shown to co-operate with c-Myc by stabilizing its protein product in 8q24 gain cancers.
• Long noncoding RNAs are a class of RNA transcripts which are longer than 200 nucleotides, evolutionarily conserved, and devoid of protein-coding potential.
• IncRNAs are frequently deregulated in various tumors and exert multiple functions in a wide range of biological processes, such as proliferation, apoptosis, cell cycle arrest, cell migration and invasion.
• PVT1 was originally identified as a cluster of breakpoints for viral integration and translocation in T- and B-cell lymphomas.
• the PVT1 locus is syntenically conserved between the human and mouse.
• PVT1 is a mutational hotspot and frequently overexpressed in cancers, its role in tumorigenesis is poorly understood.
• the dependency of MYC-driven cancer cells on PVT1 was examined.
• the driver mutation in the colorectal cancer cell line HCT116 is a mutant b-catenin gene.
• a stable b- catenin protein recruits TCF4 to upregulate MYC transcription in these cells.
• PVT1 was deleted in these cells.
• / J 17/ -deficient HCT116 cells are impaired in their tumorigenic potential compared to their wild-type controls.
• multiple lines of evidence suggest that PVT1 plays a crucial role in augmenting MYC protein in 8q24 gain cancers.
• Micro-peptides are polypeptides with a length of less than 100-150 amino acids that are encoded by short open reading frames (sORFs). In this respect, they differ from many other active small polypeptides, which are produced through the posttranslational cleavage of larger polypeptides. In terms of size, micro-peptides are considerably shorter than "canonical" proteins, which have an average length of 330 and 449 amino acids in prokaryotes and eukaryotes, respectively. Micro-peptides lack an N-terminal signaling sequences, suggesting that they are likely to be localized to the cytoplasm.
• micro-peptides have been found in other cell compartments, as indicated by the existence of transmembrane micro-peptides. They are found in both prokaryotes and eukaryotes.
• the sORFs from which micro-peptides are translated can be encoded in 5' UTRs, small genes, or polycistronic mRNAs.
• micropeptides that can inhibit Myc expression.
• the micropeptides of the present disclosure are encoded by the PVT1 locus.
• the micropeptides of the present disclosure are encoded by a splice variant of PVT 1 locus.
• the micropeptides of the present disclosure are encoded by a splice variant of PVT 1 locus, designated as PVT 1 217.
• HNB is a micro-peptide comprising an amino acid sequence MKTQLGAVKGFLHV (SEQ ID NO:l).
• HNB is a micro-peptide comprising an amino acid sequence that is 80% identical to SEQ ID NO: 1.
• the HNB polypeptide comprises a sequence that fits between the dimer interface of KRAS.
• the HNB polypeptide comprises a sequence that binds at the region of amino acid residues 147 to 156 of KRAS. In some aspects, the HNB polypeptide binds at the region of amino acid residues KTRQGVDDAF (SEQ ID NO:2) of KRAS.
• the HNB polypeptide binds part of the region of amino acid residues 147 to 156 of KRAS, such as from 148 to 156, from 149 to 156, from 150 to 156, from 151 to 156, from 152 to 156, from 153 to 156, from 154 to 156, from 155 to 156, from 147 to 155, from 147 to 154, from 147 to 153, from 147 to 152, from 147 to 151, from 147 to 150, from 146 to 149, from 147 to 148, from 154 to 155, from 154 to 156, from 153 to 154, from 153 to 155, from 152 to 156, or from 151 to 156.
• HNB is a micro-peptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:l. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. In some embodiments,
• HNB is a micro-peptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:l. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 96% identical to SEQ ID NO: 1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 97% identical to SEQ ID NO: 1. In some embodiments, HNB is a micro-peptide comprising an amino acid sequence that is 98% identical to SEQ ID NO: 1. In some embodiments, HNB is a micro peptide comprising an amino acid sequence that is 99% identical to SEQ ID NO: 1.
• the HNB polypeptides disclosed herein comprise SEQ ID NO:l with at least one conservative amino acid substitution. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO: 1 with at least two conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO: 1 with at least three conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO: 1 with at least four conservative amino acid substitution. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:l with at least five conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:l with at least six conservative amino acid substitutions.
• the HNB polypeptides disclosed herein comprise SEQ ID NO:l with at least seven conservative amino acid substitution. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO:l with at least eight conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO: 1 with at least nine conservative amino acid substitutions. In some embodiments, the HNB polypeptides disclosed herein comprise SEQ ID NO: 1 with at least ten conservative amino acid substitutions. In some embodiments, disclosed herein are HNB polypeptides comprising SEQ ID NO:l with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions in SEQ ID NO: 1. In one aspect, the micropeptide of the present disclosure can be used for a therapeutic or pharmaceutical composition for treating cancer.
• the HNB polypeptides disclosed herein comprise HNB fusion proteins.
• HNB fusion proteins comprise a HNB polypeptide and a heterologous polypeptide.
• the heterologous polypeptide is selected from the group consisting of calmodulin, polyglutamine, E-tag, FLAG, HA, His, Myc, S-tag, SBP- tag, Softag 1, Softag3, Strep-tag, TC-tag, V5, VSV, Xpress, Isopeptag, SpyTag, SnoopTag, BCCP, GST, GFP, Halo-tag, MBP, Nus-tag, Thioredoxin, albumin, an antibody, Fc domain, and combinations thereof.
• the heterologous polypeptide is an Fc domain. In some embodiments, the heterologous polypeptide targets the HNB fusion protein to a specific cell or tissue. In some embodiments, the heterologous polypeptide is fused to the N-terminus of the HNB polypeptide. In some embodiments, the heterologous polypeptide is at the C-terminus of the HNB polypeptide.
• the HNB fusion proteins comprise a HNB polypeptide and an exemplary sequence of a heterologous protein provided in Table 1 below:
• the HNB fusion polypeptides disclosed herein employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
• the HNB fusion polypeptides disclosed comprise immunologically active domains, such as an antibody epitope, to facilitate purification of the HNB fusion polypeptide. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
• Other useful fusions include linking of functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals or transmembrane regions.
• Additional HNB fusion polypeptides as disclosed herein can comprise a cell-penetrating peptide linked to a polypeptide to promote uptake of the polypeptide by the cell.
• polynucleotides encoding any one of the above HNB polypeptides.
• modified polypeptides comprising any one of the above HNB polypeptides.
• the modification is selected from the group consisting of a glycosylation and a phosphorylation.
• the modification is selected from the group consisting of: myristoylation, palmitoylation, isoprenylation, glypiation, lipolation, acylation, akylation, amidation, phosphorylation, glycation, biotinylation, pegylation, sumoylation, ubiquitination, neddylation, or pupylation.
• Modifications also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms.
• Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond.
• compositions comprising any one of the above HNB fusion polypeptides, the HNB polynucleotides, or any one of the above modified polypeptides, and an excipient.
• the excipient comprises at least one of the group consisting of maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and polyoxyethylene-sorbitan monooleate.
• the composition comprises an additional therapeutic agent.
• the additional therapeutic agent is a chemotherapeutic.
• the cancer is a MYC driven cancer. In some embodiments, the cancer is a KRas driven cancer. In some embodiments, the cancer is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers (e.g., Kaposi Sarcoma, Lymphoma, etc.), Anal Cancer,
• ALL Acute Lymphoblastic Leukemia
• AML Acute Myeloid Leukemia
• Adrenocortical Carcinoma e.g., Kaposi Sarcoma, Lymphoma, etc.
• Anal Cancer e.g., Kaposi Sarcoma, Lymphoma, etc.
• Appendix Cancer Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer (Extrahepatic), Bladder Cancer, Bone Cancer (e.g., Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma, etc.), Brain Stem Glioma, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma, etc.), Breast Cancer (e.g., female breast cancer, male breast cancer, childhood breast cancer, etc.), Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (e.g., Childhood, Gastrointestinal, etc.), Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System (e.g., Atypical Teratoid
• Lung Cancer e.g., Non-Small Cell, Small Cell, etc.
• Lymphoma e.g., AIDS-Related, Burkitt, Cutaneous T-Cell, Hodgkin, Non-Hodgkin, Primary Central Nervous System (CNS), etc.
• Macroglobulinemia e.g., Waldenstrom, etc.
• Male Breast Cancer Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia (e.g
• Parathyroid Cancer Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma,
• Prostate Cancer Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (e.g., Ewing, Kaposi, Osteosarcoma, Rhabdomyosarcoma, Soft Tissue, Uterine, etc.), Sezary Syndrome, Skin Cancer (e.g., Childhood, Melanoma, Merkel Cell Carcinoma, Nonmelanoma, etc.), Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer (e.g., with Occult Primary, Metastatic, etc.), Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ure
• the carcinoma is selected from the group consisting of acinar carcinoma , acinic cell carcinoma, acinous carcinoma, adenocystic carcinoma , adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, adrenocortical carcinoma, alveolar carcinoma, ameloblastic carcinoma, apocrine carcinoma, basal cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, cholangiocellular carcinoma, chorionic carcinoma, clear cell carcinoma, colloid carcinoma, cribriform carcinoma, ductal carcinoma in situ, embryonal carcinoma, carcinoma encuirasse, endometrioid carcinoma, epidermoid carcinoma, carcinoma ex mixed tumor, carcinoma ex pleomorphic adenoma, follicular carcinoma of thyroid gland, hepatocellular carcinoma, carcinoma in situ, intraductal carcinoma, Hurthle cell carcinoma, inflammatory carcinoma of the breast, large cell carcinoma, invasive lobular carcinoma, lobular carcinoma, lobular carcinoma in situ, intraductal
• the treatment reduces at least one symptom of a cancer.
• the treatment (a) prevents the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and/or symptom(s).
• Those in need of treatment can include those already inflicted (e.g., those with cancer, e.g. those having tumors) as well as those in which prevention is desired (e.g., those with increased susceptibility to cancer; those with cancer; those suspected of having cancer; etc.).
• cell expressing HNB polypeptides, including HNB fusion polypeptides as disclosed herein.
• cell is a mammalian cell.
• the cell is an insect cell.
• the cell is a yeast cell.
• the cell is a bacterial cell.
• Examples of cells for expressing the HNB fusion polypeptides disclosed herein include, but are not limited to, a CHO cell, a ExpiCHO-S cell, a CHO DG44 cell, a CHO-K1 cell, a myeloma cell, a hybridoma cell, a NS0 cell, a GS-NSO cell, aHEK293 cell, aHEK293T cell, aHEK293E cell, aHEK293-6E cell, a HEK293F cell, and a per.C6 cell.
• the cell is a CHO cell.
• the cell is a myeloma cell.
• the cell is selected from the group consisting of an E. coli cell, a P. mirabilis cell, a P. putidas cell, a B. brevis cell, a B. megaterium cell, a B. subtilis cell, a L. paracasei cell, a S. lividans cell, a Y. lipolytica cell, a K. lactis cell, a P. pastoris cell, a S. cerevisiae cell, a A. niger var. awamori cell, a A. oryzae cell, a L. tarentolae cell, a T. ni larvae cell, a S. frugiperda cell, a Drosophila S2 cell, a S. frugiperda SF9 cell, a T. ni cell, and a SfSWT-1 mimic cell Micropeptide Therapeutics
• therapeutic compositions comprising a peptide or a polypeptide that comprises a micropeptide, having an sequence of amino acids that is at least 80% identical to the sequence MKTQLGAVKGFLHV (SEQ ID NO: 1).
• the therapeutic composition can be a vaccine, a prophylactic, or combined with other therapeutics for treating cancer.
• the therapeutic comprising a peptide or a polypeptide that is less than 30 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 20 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 19 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 18 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1.
• the therapeutic comprising a peptide or a polypeptide that is less than 17 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 16 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 15 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1. In some embodiments, the therapeutic comprising a peptide or a polypeptide that is less than 14 amino acids long comprising an amino acid sequence of the micropeptide of SEQ ID NO: 1.
• the micropeptide comprises at least 1 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 2 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 3 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 4 conservative amino acid substitution within the sequence of SEQ ID NO: 1. In some embodiments, the micropeptide comprises at least 5 conservative amino acid substitution within the sequence of SEQ ID NO: 1.
• the micropeptide is further conjugated with one or more biomolecules.
• the biomolecule is a peptide.
• the biomolecule is a lipid.
• the micropeptide is further modified.
• the modification is selected from the group consisting of: myristoylation, palmitoylation, isoprenylation, glypiation, lipolation, acylation, alkylation, amidation, phosphorylation, glycation, biotinylation, pegylation, sumoylation, ubiquitination, neddylation, or pupylation.
• the peptide is associated with a carrier molecule.
• the carrier molecule is a lipid.
• the pharmaceutical composition described herein is for use in preparing a medicament for the treatment of a cancer in a subject.
• the selection of peptides can be guided by the given tissue to avoid side effects.
• the selection may be dependent on the specific type of cancer, the status of the disease, earlier treatment regimens, the immune status of the patient, and, of course, the HLA-haplotype of the patient.
• the vaccine according to the disclosure can contain individualized components, according to personal needs of the particular patient. Examples include varying the amounts of peptides according to the Myc expression in the particular patient, unwanted side- effects due to personal allergies or other treatments, and adjustments for secondary treatments following a first round or scheme of treatment.
• the pharmaceutical composition comprises about 1-50,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1- 40,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-30,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-20,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-10,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-5,000 ug of the micro-peptide. In some embodiments, the pharmaceutical composition comprises about 1-1,000 ug of the micro-peptide.
• compositions described herein comprise a nucleic acid that encodes a peptide or a polypeptide comprising a sequence that is at least 80% identical to SEQ ID NO: 1.
• the nucleic acid is DNA.
• the nucleic acid is RNA.
• the nucleic acid is messenger RNA (mRNA).
• the nucleic acid is an mRNA comprising a sequence encoding a sequence comprising MKTQLGAVKGFLHV (SEQ ID NO:l).
• the mRNA comprises a sequence encoding a peptide that is at least 80% identical to SEQ ID NO: 1.
• the mRNA comprises a sequence:
• the mRNA comprises more than one copy of the sequence encoding a peptide having the sequence denoted in SEQ ID NO: 1.
• the mRNA comprises a concatemer of a series of 2, 3, 4, 5, 6, 7, 8, 9, 10,
• the concatemer comprises sequences for self-cleavahle elements such as P2A, T2A or E2A in between two sequences each encoding the SEQ ID NO: 1 or a peptide sequence that is at least 80% identical to SEQ ID NO:
• mRNA processing comprises the addition of a “cap” on the N-terminal (5') end, and a “tail” on the C-terminal (3') end.
• a typical cap is a 7- methylguanosine cap, which is a guanosine that is linked through a 5 '-5 '-triphosphate bond to the first transcribed nucleotide. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
• the tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3' end of the mRNA molecule. The presence of this “tail” serves to protect the mRNA from exonuclease degradation. mRNA is translated by the ribosomes into a series of amino acids that make up a protein.
• mRNAs according to the present disclosure may be synthesized according to any of a variety of known methods.
• mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT).
• IVT in vitro transcription
• IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
• RNA polymerase e.g., T3, T7 or SP6 RNA polymerase
• the present disclosure may be comprise a therapeutic or pharmaceutical composition comprising synthesized mRNA of a variety of lengths.
• the present disclosure comprise in vitro synthesized mRNA of or greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length.
• the present disclosure may comprise a therapeutic composition comprising a synthesized mRNA ranging from about 1- 20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length.
• typical mRNAs may be about 1 kb to about 5 kb in length. More typically, the mRNA will have a length of about 1 kb to about 3 kb. However, in some embodiments, the mRNA in the composition of the disclosure is much longer (greater than about 20 kb).
• the present disclosure may comprise mRNA containing one or more modifications that typically enhance stability.
• one or more modifications are selected from modified nucleotide, modified sugar phosphate backbones, 5' and/or 3' untranslated region.
• mRNAs are modified to enhance stability.
• Modifications of mRNA can include, for example, modifications of the nucleotides of the mRNA.
• a modified mRNA according to the disclosure can thus include, for example, backbone modifications, sugar modifications or base modifications.
• antibody encoding mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
• nucleotide analogues modified nucleotides
• purines adenine (A), guanine (G)
• pyrimidines thymine (T), cytosine (C), uracil (U)
• modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
• mRNA synthesis includes the addition of a “cap” on the N-terminal (5') end, and a “tail” on the C-terminal (3') end.
• the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
• the presence of a “tail” serves to protect the mRNA from exonuclease degradation.
• mRNAs include a 5' cap structure.
• a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5 '5 '5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
• GTP guanosine triphosphate
• cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
• mRNAs include a 5' and/or 3' untranslated region.
• a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element.
• a 5' untranslated region may be between about 50 and 500 nucleotides in length.
• a 3 ' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs.
• a 3 ' untranslated region comprises a poly A tail, that may be between 50 and 500 nucleotides in length or longer.
• the poly A tail may be 50-250 nucleotides long.
• the therapeutic or pharmaceutical composition of the present disclosure is delivered to a subject in need thereof, wherein the therapeutic or pharmaceutical composition comprises a peptide, a polypeptide or a nucleic acid encoding the peptide or polypeptide, wherein the peptide or the polypeptide comprises a sequence as set forth in SEQ ID NO: 1, or a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 1.
• the nucleic acid can be delivered directly, as "naked DNA”, or “naked mRNA”.
• the nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5.204,253, which is herein incorporated by reference for purposes of describing ballistic delivery administration.
• Particles comprised solely of DNA can be administered.
• DNA can be adhered to particles, such as gold particles.
• the nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids.
• Lipid- mediated gene delivery methods are described, for instance, in Mannino & Gould-Fogerite , BioTechniques 6(7): 682-691 (1988); U.S. Pat No. 5,279,833; Feigner et ah, Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987) and others, which are hereby incorporated by reference for purposes of describing lipid-mediated gene delivery methods.
• pharmaceutical formulations comprising the HNB polypeptides disclosed herein are made to be compatible with a particular local, regional or systemic administration or delivery route.
• pharmaceutical formulations include carriers, diluents, or excipients suitable for administration by particular routes.
• routes of administration for compositions herein are parenteral, e.g., intravenous, intra-arterial, intradermal, intramuscular, subcutaneous, intra-pleural, transdermal (topical), transmucosal, intra-cranial, intra-spinal, intra-ocular, rectal, oral (alimentary), mucosal administration, and any other formulation suitable for the treatment method or administration protocol.
• pharmaceutical solutions or suspensions for parenteral application include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• pH is adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• compositions for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N. J.), or phosphate buffered saline (PBS).
• the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), or suitable mixtures thereof.
• Fluidity is maintained, in some embodiments, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
• Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.
• Isotonic agents for example, sugars; polyalcohols such as mannitol or sorbitol; or sodium chloride, in some embodiments, are included in the composition.
• an agent which delays absorption in some embodiments, for example, aluminum monostearate or gelatin prolongs absorption of injectable compositions.
• sterile injectable formulations are prepared by incorporating the HNB polypeptides disclosed herein in the required amount in an appropriate solvent with one or more of the above ingredients.
• dispersions are prepared by incorporating the HNB polypeptides disclosed herein into a sterile vehicle containing a basic dispersion medium and any other ingredient.
• methods of preparation include, for example, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously prepared solution thereof.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• transmucosal administration is accomplished through the use of nasal sprays, inhalation devices (e.g., aspirators) or suppositories.
• the active compounds are formulated into ointments, salves, gels, creams or patches.
• the pharmaceutical formulations are prepared with carriers that protect against rapid elimination from the body, such as a controlled release formulation or a time delay material such as glyceryl monostearate or glyceryl stearate.
• the formulations in some embodiments, are also delivered using articles of manufacture such as implants and microencapsulated delivery systems to achieve local, regional or systemic delivery or controlled or sustained release.
• nanoparticle-polypeptide complexes comprising a HNB polypeptide as disclosed herein in association with a nanoparticle, wherein the HNB polypeptide is modified by the addition of a chemical moiety that facilitates cellular uptake of the complex.
• the nanoparticle may be a lipid-based nanoparticle, a superparamagnetic nanoparticle, a nanoshell, a semiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle, a silicon-based nanoparticle, a silica-based nanoparticle, a metal- based nanoparticle, a fullerene or a nanotube.
• the nanoparticle may be a lipid-based nanoparticle.
• the lipid-based nanoparticle may be a liposome, a neutral liposome, a DOPC liposome or a DOTAP xholesterol vesicle.
• the liposome may be a DOPC liposome.
• liposomes take advantage of the increased fenestrations in the cancer neo vasculature to enhance liposome concentration at tumor sites.
• the nanoparticle is a superparamagnetic nanoparticle. Superparamagnetic nanoparticles ranging in diameter from about 10 to 100 nm are small enough to avoid sequestering by the spleen, but large enough to avoid clearance by the liver.
• the nanoparticle is a superparamagnetic nanoparticle, and the nanoparticle-polypeptide complex is within a liposome or a DOTAP: cholesterol vesicle.
• the liposome may be a DOPC liposome.
• the chemical moiety may be a fatty acid (e.g., a C4-C18 fatty acid, stearate or myristate).
• the chemical moiety may be a cell penetrating peptide.
• the cell penetrating peptide may be derived from HIV Tat, herpes virus VP22, or the Drosophila Antennapedia homeobox gene product.
• the HNB polypeptides can be targeted to specific tissues and cells.
• the nanoparticle-polypeptide complexes comprising a HNB polypeptide as disclosed herein can be conjugated to a cell targeting moiety.
• the targeting moiety can be, but is not limited to, a protein, peptide, lipid, steroid, sugar, carbohydrate or synthetic compound.
• Cell targeting moieties such as ligands recognize and bind to their cognate receptors on the surface of cells.
• an antibody can act as cell targeting moieties by recognizing a cognate antigen on a cell surface.
• Targeted nanoparticle-polypeptide complexes can enhance the specificity of disease treatment and increase the amount of therapeutic agent entering a targeted cell.
• Liposomes can be variously used to deliver nucleic acids or peptides.
• the peptides disclosed herein may also be administered via liposomes, which target the peptides to a particular cells tissue, such as lymphoid tissue. Liposomes are also useful in increasing the half- life of the peptides. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome.
• liposomes filled with a desired peptide of the disclosure can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic /immunogenic peptide compositions.
• Liposomes for use in the disclosure are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng.
• a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
• Therapeutically effective amounts or dosages of the HNB polypeptides disclosed herein and pharmaceutical formulations comprising the HNB polypeptides disclosed herein are contemplated to include dosages of 0.01 mg to 20 mg, for example, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.1 mg, 4.2 mg, 4.3
• Therapeutically effective amounts or dosages are contemplated to include dosages of 0.1 mg to 2.0 mg.
• the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
• aqueous carriers may be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
• concentration of peptides of the disclosure in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
• compositions include “pharmaceutically acceptable” and “physiologically acceptable” carriers, diluents or excipients.
• pharmaceutically acceptable and “physiologically acceptable” include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration to a mammal, for example a human.
• such formulations are contained in a liquid, e.g., emulsion, suspension, syrup or elixir; or solid form, i.e., tablet (e.g., coated or uncoated, immediate, delayed, continuous, or pulsatile release), capsule (e.g., hard or soft, immediate, delayed, continuous, or pulsatile release), powder, granule, crystal, or microbead.
• a liquid e.g., emulsion, suspension, syrup or elixir
• solid form i.e., tablet (e.g., coated or uncoated, immediate, delayed, continuous, or pulsatile release), capsule (e.g., hard or soft, immediate, delayed, continuous, or pulsatile release), powder, granule, crystal, or microbead.
• supplementary compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
• supplementary compounds are also incorporated into the formulations.
• kits for treating a subject in need thereof comprising administering the subject a pharmaceutical composition comprising a peptide comprising an amino acid sequence set forth in SEQ ID NO: 1, or a sequence that is at least 80% identical to SEQ ID NO: 1.
• kits for treating a subject in need thereof comprising administering the subject a pharmaceutical composition comprising a nucleic acid encoding a peptide comprising an amino acid sequence set forth in SEQ ID NO: 1, or a sequence that is at least 80% identical to SEQ ID NO: 1.
• compositions comprising the peptides disclosed herein may be administered to an individual already suffering from cancer.
• Pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
• the compositions may be administered at the site of surgical excision to induce a local immune response to the tumor.
• compositions for parenteral administration which comprise a solution of the peptides and vaccine compositions are dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
• nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and others.
• a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the disclosure, and more preferably at a concentration of 25%- 75%.
• the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant.
• Typical percentages of peptides are 0.01 %- 20% by weight, preferably 1%-10%.
• the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
• Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
• Mixed esters, such as mixed or natural glycerides may be employed.
• the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant.
• a carrier can also be included as desired, as with, e.g., lecithin for intranasal delivery.
• HNB polypeptides disclosed herein may also be combined with therapy designed to eliminate cancer cells present in a tumor, e.g. in a combination therapy with various other conventional methods of treating cancer cells, for example chemotherapy, radiation therapy, etc.
• Chemotherapeutic agents may include, for example, temozolomide, protein-bound paclitaxel, romidepsin.
• cyclophosphamide vincristine, doxorubicin, methotrexate, ifosfamide, etoposide, and cytarabine (CODOX-M/IVAC) plus rituximab; rituximab plus etoposide, prednisone, vincristine (Oncovin), and doxorubicin (R- EPOCH) and rituximab plus cyclophosphamide, vincristine, doxorubicin (Adriamycin), and dexamethasone (R-Hyper CVAD).
• the chemotherapeutic agent is selected from the group consisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afmitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Apr
• Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A (Recombinant Interferon Alfa-2b), Iodine 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kyprolis
• methods of preventing PVT1 chromosomal fusions are contemplated.
• mechanisms of suppressing the chromosomal breakage and repair machinery of a cell in a spatio-temporal manner guided to the PVT1 locus is contemplated.
• mechanisms for suppression of inter-chromosomal fusion involving the PVT1 locus is contemplated.
• mechanisms for suppression of intra-chomosomal fusion fusion involving the PVT1 locus is contemplated.
• mechanisms for preventing extrachromosomal DNA formation fusion involving a nucleic acid sequence found within the PVT1 locus is contemplated.
• An extrachromosomal DNA formed as a result of breakage at or within the PVT1 locus may be 10, 20, 30, 40, 50, 100, 1000, 10,000 nucleotides in length.
• An inhibitory RNA mediated decay of an extrachromosomal nucleic acid (ecDNA) comprising the 5’ region of PVT-1 may be contemplated herein.
• ecDNA extrachromosomal nucleic acid
• a site -directed or sequence-targeted approach to prevent formation of extrachromosomal DNA comprising the PVT-1 locus may be contemplated.
• Example 1 Identification of a tumor suppressor splice variant PVT1 217 [0106] A previously undescribed PVT1 splice variant that can regulate Myc protein was identified. Using the ENSEMBL database, PVT1 splice variants were analyzed. siRNAs were designed to target specific PVT1 splice variants and tested on MSTO-211H cell line for alteration in Myc expression levels.
• siRNA dubbed Ex9 increased the expression of Myc protein in the MSTO-211H cell line compared to control siRNA (si ctrl) or compared to si_C2 (an siRNA specific for CircPVTl, where CircPVTl is a splice variant previously reported to upregulate Myc expression) (FIG. 1).
• si_E9 knocked down the expression of a PVT1 splice variant, PVT1 217, indicating a role for PVT1 217 in regulating Myc levels.
• PVT 1 217 (referred to herein as PVTl ts ) comprises 4 exons as shown in the Ensembl database, but its 4 th exon is incompletely annotated in the Ensembl database.
• the correct genomic co-ordinates of PVTl ts are depicted in FIG 3.
• Example 2 PVTlts regulates cell proliferation and Myc expression in Hi- and Lo-Myc cell lines.
• si_E9 Small inhibitory RNA was then generated against the PVTl ts splice variant.
• the si_E9 was shown to target a unique sequence in the exon 4 of PVTl ts (FIG. 4A).
• si_E9 significantly knocks down PVT Its while expression of the other transcripts in the PVT1 locus remain unaffected (data not shown).
• siRNA mediated depletion of PVT Its was carried out in Hi- and Lo-Myc cell lines, which resulted in increased proliferation of the Hi- as well as Lo- Myc cell lines (FIG. 4B). Additionally, western blot analysis of Myc protein was performed in these cell lines.
• Micro-peptide HNB can affect cell growth and proliferation and regulates Myc protein expression in Hi-and Lo-Myc cell lines.
• HNB Hi-and Lo- Myc cell lines were transfected with lentiviral vectors expressing GFP, HNB or HNB where the start site has been mutated (HNB(ATG>TGA)). All of the 8 cell lines where HNB is overexpressed (but not the ones which are transfected with GFP or HNB(ATG>TGA)) failed to proliferate, suggesting a role for HNB as a tumor suppressor (FIG. 7A and 7B).
• an inducible lentiviral system in which the transgene could be expressed under a Doxycycline inducible promoter.
• the system was verified by inserting eGFP as the transgene and by flow analysis of GFP + cells in absence and presence of doxycycline as shown in FIG. 8.
• Two stable cell lines MSTO-211H-GFP 1 and U20S-GFP 1 were created where a GFP transgene was placed under the regulation of a Dox inducible promoter showed robust expression of GFP following treatment of lug/ml of Doxycycline within 24 hours.
• Example 5 Micro-peptide HNB is predicted to inhibit the dimerization of KRAS oncoprotein.
• HNB may act as a tumor suppressor
• a computational analysis was carried out using the predicted structure of HNB (FIG. 10) and its possible interacting partners.
• Micropeptides are usually single domain peptides.
• Deepview Swiss-Pdb Viewer
• This application provides a user-friendly interface to analyze multiple proteins and their possible interactions at the same time (https://spdbv.vital-it.ch).
• RNA-seq transcriptomic studies
• CCLE cancer cell line encyclopedia
• PVT1 was also the most promiscuous of fusion-genes, partnering with at least 54 different fusion partners indicating that PVT1 is a nodal locus in tumorigenesis and there are diverse ways in which this locus is disrupted in cancer (FIG. 15).
• PVT1 gene-fusions are formed as extrachromosomal double minutes (dmin) or extrachromosomal DNA (ecDNA). If such ecDNAs are formed from the juxtaposition of the PVT1 5’ end to genes centromeric (upstream) to PVT1, then it would lead to the enrichment of the PVT1 5’ region, relative to its 3’ end which is not involved in the ecDNA formation.
• dmin extrachromosomal double minutes
• ecDNA extrachromosomal DNA
• COLO-320DM colonal
• SK-PN-DW primary neuroectodermal tumor (PNET)
• D458 Medulloblastoma
• Cell lines that are copy number neutral for the MYC- PVT1 region and do not harbor any breakpoint in the PVT1 locus - U20S (osteosarcoma), BxPC-3 (pancreatic adenocarcinoma) and DU 145 (prostate adenocarcinoma) were chosen as control.
• D458 appeared to contain a mix of homology stained regions (hsr) as well as ecDNAs with excess representation of 5’- half of PVT1.
• hsr homology stained regions
• ecDNAs with excess representation of 5’- half of PVT1.
• We confirmed the ecDNA junctions by reconstructing the paired-end discordant junctional reads in silico in COLO-320DM and D458 and confirmed their presence using PCR followed by Sanger sequencing (FIG. 20A- 20B).

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